The bone morphogenetic protein (BMP) family belongs to the transforming growth factor-β (TGF-β) superfamily and includes a group of closely related polypeptides identified initially by their capacity to stimulate ectopic bone formation in vivo. BMPs are synthesized as large precursor proteins before being processed and proteolytically cleaved to form mature carboxyl-terminal dimers. BMP members, such as BMP1 to BMP14, have been known to have different levels of bone morphogenetic activity. For example, BMP2 and BMP4, which are expressed by osteoblasts as they differentiate, have been shown to stimulate osteoblast differentiation and bone nodule formation in vitro. In addition, recombinant BMP2 and BMP4 can induce new bone formation when injected locally into the subcutaneous tissues of rats.
BMPs transduce signals through binding cooperatively to both Type I and Type II receptors, which are trans-membrane serine-threonine kinase receptors. Transphosphorylation of the Type I receptor by the Type II kinase in the cytoplasmic domain triggers a downstream signaling cascade. However, little is known about signal transduction involved in BMP signaling pathways. Effectors (e.g., Mad proteins), which responded downstream to BMP signals, have recently been found in human and Xenopus tissues.
Bone morphogenetic protein 4 (BMP4) is a member of the BMP family and, like other BMPs, is a multifunctional regulator during vertebrate development. BMP4 has been shown to play important roles in the establishment of the basic embryonic body plan (e.g., mesoderm formation, left-right asymmetry, dorsal-ventral patterning in vertebrates), in morphogenesis (e.g., skeletal development and limb patterning), and in the development of organs and tissues (e.g., the development of kidney, lung, heart, teeth, gut, and skin, and formation of the central and peripheral nervous system, etc.). In fact, the expression of the bone morphogenetic proteins and their receptors has been identified in a large variety of cells, tissues, and organs, and in specific temporal and spatial patterns.
Mechanisms regulating the expression of bmp genes in vivo are still largely unknown despite the identification of two mouse BMP4 transcripts and cloning of a mouse BMP4 gene. In addition, two human BMP4 transcripts have been identified and two human BMP4 promoter regions have been cloned. The two mouse BMP4 transcripts result from two alternative 5′non-coding exons, 1A and 1B in the BMP4 promoter region. It was found that 1A promoter is primarily utilized in bone cell cultures, and a chicken ovalbumin upstream-Transcription Factor I (coup-TFI) was demonstrated in vitro to negatively regulate murine BMP4 1A promoter in fetal rat calvariae cells. Further, various transcripts resulting from several promoters have been observed for a BMP4 homologue in Drosophila melanogaster, decapentaplegic protein (dpp). The use of diverse and separate promoter regions for one BMP4 gene in different cells derived from different tissues suggests a cell-specific or tissue-specific regulation of BMP4 gene expression. Given the unstable half-life of most BMP4 transcripts, expression of bmp genes is largely regulated at the transcriptional level.
Although considerable efforts have been focused on the study of BMP4 function during zebrafish development, the molecular mechanisms regarding the expression of zebrafish BMP4 remain unclear. In contrast to human and murine BMP4, a single transcript has so far been identified for the zebrafish BMP4 gene. The finding and the materials and methods disclosed in the present invention suggest promoter structure, intron/exon organization, and cell-specific and/or tissue-specific regulation of zebrafish BMP4 gene expression are different from human and murine BMP4 despite high level of amino acid sequence homology among BMP4 proteins from humans, mice, and zebrafish.
Therefore, there is a need to understand the regulation of zebrafish BMP4 expression, to provide further insights into molecular mechanisms, regulatory DNA sequences, and transcription factors that regulate development of various BMP4-expressing tissues and organs, and to identify molecular compounds/substances that induce or inhibit the expression of zebrafish BMP4 expression.
Recently, transgenic technology using various reporter genes, e.g., green fluorescent protein (GFP), has provided a powerful means to study gene function and the regulation of gene expression. Thus, there is a need to provide cell lines and transgenic fish to allow real-time imaging of various morphogenetic processes in different cells, organs, tissues, and during embryogenesis.